Telomeres are specialized structures present at the ends of linear eukaryotic chromosomes that function in replicating and maintaining the integrity of the chromosomal ends. Telomeres distinguish the chromosomal terminus from other types of double stranded breaks responsible for initiating cell growth arrest and aberrant chromosomal fusions.
Telomeric sequences vary between species, but their essential features are similar between eukaryotes. The telomeric DNA is generally composed of tandem repeats of a basic sequence unit. Telomeric repeats of some organisms are perfect repeats, such as sequence TTAGGG seen in humans or slime mold. Others, such as those of yeast or protozoans, have irregular repeat sequences. In general, the G rich strand runs 5′ to 3′ to the chromosomal terminus. The length of the repeat sequences range from a few to tens of kilo base pairs.
In some organisms, the characteristic telomeric repeat sequences are absent and are substituted by other sequences that function similar to telomere repeat sequences. For example, in Drosophila melanogaster, the telomeres are generally composed of non-LTR—type retrotransposons, called HeT-A and TART elements. In the mosquito Anopheles gambiae, the chromosomal ends are composed of arrays of complex sequence tandem repeats rather than short repeat sequences.
In some organisms, a 3′ single stranded overhang is present at the terminus of the telomere. The length of the single stranded region is variable, extending from about 100 bp or more. Under defined conditions in vivo or in vitro, the overhanging terminus associates with various telomere associated proteins and invades the double stranded telomere region to form t-loop structures (Griffith, J. D. et al., Cell 97: 503-514 (1999); Munoz-Jordan et al., EMBO J. 20: 579-588 (2001)). Formation oft-loop structures is believed to function in negatively regulating telomere elongation by telomerase, and in addition, provide a mechanism for sequestering the single stranded ends to protect them from degradation and to suppress activation of DNA damage checkpoint pathways.
Typically, the DNA replicative machinery acts in the 5′ to 3′ direction, and synthesis of the lagging strand occurs discontinuously by use of short RNA primers that are degraded following strand synthesis. Since sequences at the 3′ end of a linear DNA are not available to complete synthesis of the region previously occupied by the RNA primer, the terminal 3′ region of the linear chromosome is not replicated. This “end replication problem” is solved by the action of telomerase, a telomere specific ribonucleoprotein reverse transcriptase. The telomerase enzyme has an integral RNA component that acts as a template for extending the 3′ end of the telomere. Repeated extensions by telomerase activity results in the generation of telomere repeats copied from the telomerase-bound RNA template. Following elongation by telomerase, lagging strand synthesis by DNA polymerase completes formation of the double stranded telomeric structure.
In normal human somatic cells, telomerase is not expressed or expressed at low levels. Consequently, telomeres shorten by 50-200 bp with each cell division until the cells reach replicative senescence, at which point the cells loose the capacity to proliferate. This limited capacity of cells to replicate is generally referred to as the Hayflick limit, and may provide cells with a counting mechanism, i.e., a mitotic clock, to count cell divisions and regulate cellular development. Correspondingly, activation of telomerase in cells lacking telomerase activity, for example by expressing telomerase from a constitute retroviral promoter or activation of endogenous polymerase, allows the cells to maintain proliferative capacity and leads to immortalization of the cell.
Interestingly, these immortalized cells have short stable telomeres while the shortest telomeres become extended. This phenomena suggests that telomerase enzyme protects short telomeres from further shortening while extending those that have fallen below a certain threshold length. Thus, presence of telomerase activity does not appear to be necessary when telomeres are a certain length, but becomes critical to maintenance of telomere integrity when it falls below a critical limit.
It is well established that the length and integrity of telomeres is important for proper segregation of chromosomes and cell growth. For example, development of many types of cancers correlates with activation of telomere maintenance while cell senescence correlates with loss of telomere integrity. Shortening of telomere induced by inhibiting telomerase activity can lead to proliferative senescence and cell apoptosis (Zhang, X. et al., Genes Dev. 2388-99 (1999)). Moreover, genetic knockouts of telomerase RNA in mice results in animals with developmental defects, age related pathologies, and increased cancer susceptibility (Rudolph, K. L. et al., Cell 96: 701-12 (1999); Herrera, E. et al., EMBO J. 18: 2950-60 (1999)). Similarly, in the autosomal dominant disorder of dyskeratosis congenita (DKC), which arises from a mutation in the gene encoding the RNA component of telomerase, afflicted patients display accelerated telomere shortening and die at a median age of 16 years (maximum approximately 50 years), usually from severe infections secondary to bone marrow failure. Clinical features of DKC patients, further suggestive of accelerated aging, include premature graying and loss of hair; skin dyspigmentation; poor wound healing; high risk of severe infections; and an increased incidence of malignancies, osteoporosis, and pulmonary fibrosis. In addition, the shortest average telomere lengths measured in blood DNA from normal elderly individuals overlap with the highest average telomere lengths measured in blood from DKC patients.
In view of the role telomeres play in cell growth and cell senescence, it is desirable to have methods of predicting the occurrence of age related diseases and mortality risk based on length of telomeres. This will provide a basis for identifying individuals with increased risk of developing particular age-associated diseases, such as cancer and hypertension, such that early medical intervention can be administered to individuals in high risk groups. In addition, these methods can be used to identify genetic and environmental factors that may play a role in changing the aging process or altering disease susceptibility in individuals and in populations.